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1.
Int Heart J ; 65(5): 939-944, 2024.
Article in English | MEDLINE | ID: mdl-39343597

ABSTRACT

Right ventricular dysfunction is a key clinical issue for the viability of donation-after-circulatory-death (DCD) heart transplantation. DCD hearts with volume overload have the potential to exhibit aggravated right ventricular dysfunction following heart transplantation. The c-jun/c-fos mRNAs are genes that immediately respond to myocardial cell stretch. We assessed myocardial cell stretch during asphyxia-induced cardiac arrest by measuring c-jun/c-fos mRNA expression levels. The trachea was dissected and ligated to initiate asphyxiation in anesthetized Wistar rats under paralyzed ventilation. The hearts were harvested at 4 time points: 0, 15, 30, and 45 minutes after the termination of ventilation. Free walls of the right and left ventricles and the interventricular septum were sectioned. Total RNA was extracted from these tissues, and cDNA was synthesized using reverse transcription. The c-jun/c-fos mRNA expression levels were quantified using the droplet digital polymerase chain reaction method. In the left ventricle, c-jun/c-fos expression levels rapidly increased at 15 minutes, but the expression levels returned to the baseline level at 30 minutes after tracheal ligation. In contrast, in the right ventricle, c-jun/c-fos expression levels gradually increased and peaked 30 minutes after tracheal ligation. Myocardial cell stretching in the right ventricle is prolonged after asphyxia-induced cardiac arrest compared to that in the left ventricle, which may lead to right ventricular dysfunction after DCD heart transplantation.


Subject(s)
Asphyxia , Heart Arrest , Proto-Oncogene Proteins c-fos , RNA, Messenger , Animals , Male , Rats , Asphyxia/complications , Asphyxia/metabolism , Disease Models, Animal , Heart Arrest/metabolism , Heart Arrest/genetics , Heart Transplantation , Heart Ventricles/metabolism , Heart Ventricles/physiopathology , Myocardium/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/metabolism , Proto-Oncogene Proteins c-jun/genetics , Rats, Wistar , RNA, Messenger/metabolism , RNA, Messenger/genetics
2.
Diabetes Ther ; 2024 Sep 30.
Article in English | MEDLINE | ID: mdl-39347897

ABSTRACT

INTRODUCTION: Imeglimin is a first-in-class, novel, oral glucose-lowering agent for the treatment of type 2 diabetes mellitus. The efficacy and safety of imeglimin as an antidiabetic agent have been investigated in clinical trials. However, its metabolic effects in humans have not yet been fully elucidated. METHODS: The Study to InveStIgate the Metabolic Action of Imeglimin on patients with type 2 diabetes mellitus (SISIMAI) is a single-arm intervention study. In this study, we have recruited 25 patients with type 2 diabetes to receive 2000 mg/day imeglimin for 20 weeks. We perform a 75-g oral glucose tolerance test (OGTT) with double-glucose tracers, a two-step hyperinsulinemic-euglycemic clamp with glucose tracer, ectopic fat measurement by proton magnetic resonance spectroscopy, visceral/subcutaneous fat area measurement by magnetic resonance imaging, muscle biopsy, and evaluation of fitness level by cycle ergometer before and after imeglimin administration. PLANNED OUTCOMES: The primary outcome is the change in area under the curve of glucose levels during the OGTT after 20 weeks of imeglimin treatment. We also calculate the endogenous glucose production, rate of oral glucose appearance, and rate of glucose disappearance from the data during the 75-g OGTT and compare them between pre- and post-treatment. Additionally, we will compare other parameters, such as the changes in tissue-specific insulin sensitivity, ectopic fat accumulation, visceral/subcutaneous fat area accumulation, and fitness level between each point. This is the first study to investigate the organ-specific metabolic action of imeglimin in patients with type 2 diabetes mellitus using the 75-g OGTT with the double tracer method. The results of this study are expected to provide useful information for drug selection based on the pathophysiology of individual patients with type 2 diabetes mellitus. TRIAL REGISTRATION: jRCTs031210600.

3.
J Pathol ; 263(4-5): 429-441, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38837231

ABSTRACT

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Subject(s)
Carcinoma, Pancreatic Ductal , Cell Lineage , Pancreatic Neoplasms , Animals , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Mice , Mice, Transgenic , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cell Transformation, Neoplastic/metabolism , Islets of Langerhans/pathology , Islets of Langerhans/metabolism , Antigens, Polyomavirus Transforming/genetics , Antigens, Polyomavirus Transforming/metabolism , Gene Expression Regulation, Neoplastic , Humans
4.
Biochem Biophys Res Commun ; 712-713: 149960, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38640734

ABSTRACT

An essential ketone body, ß-hydroxybutyrate (BOHB), plays various roles in physiological regulations via protein acylations such as lysine acetylation and ß-hydroxybutyrylation. Here, to understand how BOHB systemically regulates acylations from an overarching perspective, we administered a ketogenic diet to mice to increase BOHB concentration and examined acylations. We found that global acetylation and ß-hydroxybutyrylation dramatically increase in various organs except for the brains, where the increase was much smaller than in the other organs. Interestingly, we observe no increase in histone acetylation in the organs where significant global protein acetylation occurs despite a substantial rise in histone ß-hydroxybutyrylation. Finally, we compared the transcriptome data of the mice's liver after the ketogenic diet to the public databases, showing that upregulated genes are enriched in those related to histone ß-hydroxybutyrylation in starvation. Our data indicate that a ketogenic diet induces diverse patterns of acylations depending on organs and protein localizations, suggesting that different mechanisms regulate acylations and that the ketogenic diet is associated with starvation in terms of protein modifications.


Subject(s)
3-Hydroxybutyric Acid , Diet, Ketogenic , Histones , Mice, Inbred C57BL , Animals , Histones/metabolism , Mice , 3-Hydroxybutyric Acid/metabolism , Male , Acylation , Liver/metabolism , Acetylation , Organ Specificity , Proteins/metabolism , Proteins/genetics , Transcriptome
5.
Nat Metab ; 6(3): 550-566, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38448615

ABSTRACT

The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.


Subject(s)
Lysine , Sirtuins , Mice , Animals , Lysine/chemistry , Lysine/metabolism , Ammonia , Sirtuins/metabolism , Mice, Knockout , Urea
6.
J Endocr Soc ; 8(3): bvae016, 2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38370441

ABSTRACT

Context: Older adults with sarcopenic obesity are at high risk for type 2 diabetes mellitus (T2DM). However, few East Asians have sarcopenic obesity. Since many East Asians have insulin resistance (IR) without obesity, it is possible that older East Asians with sarcopenia and IR might be at high risk for T2DM. However, this relationship has not been studied. Methods: This cross-sectional study included 1629 older adults aged 65 to 84 years registered in the Bunkyo Health Study. All underwent a 75-g oral glucose tolerance test and handgrip strength measurement. Participants were classified into 4 groups by possible sarcopenia (handgrip strength <28 kg in men and <18 kg in women) and IR status (triglyceride glucose [TyG] index ≥8.79 for men and ≥8.62 for women [third quartile]). Modified Poisson regression was used to estimate relative risk (RR) and 95% CIs for T2DM with adjustment for confounding factors. Results: The mean age was 73.1 ± 5.4 years. T2DM was diagnosed in 212 (13.0%) participants. After adjusting for age, sex, body mass index, use of lipid-lowering medications, hypertension, and cardiovascular disease, possible sarcopenia and IR were associated with T2DM, with their coexistence showing a notably stronger association (control: RR, 1.00 [Reference]; possible sarcopenia: RR, 1.55 [95% CI, 1.04-2.30]; IR: RR, 2.69 [95% CI, 1.99-3.65]; and IR possible sarcopenia: RR, 4.76 [95% CI, 3.34-6.79]). Conclusion: Possible sarcopenia based on low handgrip strength and IR based on the TyG index are independently associated with T2DM in older Japanese individuals. Their coexistence shows a particularly strong association with T2DM.

7.
Diabetes Ther ; 15(4): 883-892, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38363542

ABSTRACT

INTRODUCTION: Recent studies have shown that the quality of life (QOL) of people living with type 1 diabetes (T1D) is poor and must be improved. However, the living situation and QOL of adults living with T1D in Japan have not been fully clarified. This study will examine their lifestyle, QOL, and clinical situation, as well as the relationships between them. METHODS: This is a prospective, 5-year follow-up observational study. Between December 2019 and September 2021, we enrolled adults in Japan who were living with T1D and receiving insulin therapy, and are acquiring longitudinal clinical data and the responses to seven questionnaires regarding lifestyle and QOL. The primary study outcomes are (1) the relationship between Problem Areas in Diabetes (PAID) scores and various factors including demographic data, clinical characteristics, medical history, lifestyle habits, treatment history, biochemical data, and the scores of questionnaires; and (2) the relationship between Beck Depression Inventory (BDI)-II scores and various factors aforementioned. The secondary outcomes are the relationships between various factors aforementioned and each of the following: (1) blood glucose control, (2) blood lipid control, (3) dietary patterns, (4) fear of hypoglycemia, (5) sleep patterns, and (6) physical activity. PLANNED OUTCOME: We registered 352 participants. The median age was 49 (41-63) years, and the median duration of T1D was 13 (8-20) years. All the results will be available in 2026. We expect to clarify the factors associated with decreased QOL, and that this knowledge will contribute to improving QOL in adults in Japan who are living with T1D and receiving insulin therapy. TRIAL REGISTRATION: Clinical Trials.gov identifier, UMIN000044088.

8.
J Endocr Soc ; 8(2): bvad164, 2024 Jan 05.
Article in English | MEDLINE | ID: mdl-38188453

ABSTRACT

Context: Older adults have a high prevalence of new-onset diabetes, often attributed to age-related decreases in insulin sensitivity and secretion. It remains unclear whether both insulin sensitivity and secretion continue to deteriorate after age 65. Objective: To investigate the effects of aging on glucose metabolism after age 65 and to identify its determinants. Methods: This cross-sectional study involved 1438 Japanese older adults without diabetes. All participants underwent a 75-g oral glucose tolerance test (OGTT). Body composition and fat distribution were measured with dual-energy X-ray absorptiometry and magnetic resonance imaging. Participants were divided into 4 groups by age (65-69, 70-74, 75-79, and 80-84 years) to compare differences in metabolic parameters. Results: Mean age and body mass index were 73.0 ± 5.4 years and 22.7 ± 3.0 kg/m2. The prevalence of newly diagnosed diabetes increased with age. Fasting glucose, fasting insulin, the area under the curve (AUC)-insulin/AUC-glucose and insulinogenic index were comparable between groups. AUC-glucose and AUC-insulin during OGTT were significantly higher and Matsuda index and disposition index (Matsuda index · AUC-insulin/AUC-glucose) were significantly lower in the age 80-84 group than in the age 65-69 group. Age-related fat accumulation, particularly increased visceral fat area (VFA), and elevated free fatty acid (FFA) levels were observed. Multiple regression revealed strong correlations of both Matsuda index and disposition index with VFA and FFA. Conclusion: Glucose tolerance declined with age in Japanese older adults, possibly due to age-related insulin resistance and ß-cell deterioration associated with fat accumulation and elevated FFA levels.

9.
Diabetologia ; 67(1): 156-169, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37870650

ABSTRACT

AIMS/HYPOTHESIS: Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. METHODS: We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. RESULTS: Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. CONCLUSIONS/INTERPRETATION: Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. DATA AVAILABILITY: Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.


Subject(s)
Glucagon-Secreting Cells , Insulin-Secreting Cells , Islets of Langerhans , Mice , Animals , Glucagon/metabolism , Glucagon-Secreting Cells/metabolism , Insulin-Secreting Cells/metabolism , Cell Differentiation/genetics , Gene Expression Profiling , Islets of Langerhans/metabolism
10.
Diabetes ; 72(11): 1609-1620, 2023 Nov 01.
Article in English | MEDLINE | ID: mdl-37625131

ABSTRACT

The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic ß-cells associated with the pathogenesis of diabetes. However, it is expensive and time consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre expression mice. We establish a ßCas9 system with mice expressing Cas9 in pancreatic ß-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant ßCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in ß-cells. We also show significant ß-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to ßCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO ß-cells into glucagon-expressing cells. We successfully generated a convenient ß-cell-specific gene KO system with ßCas9 mice and AAV8-mediated gRNA delivery.

11.
J Diabetes Investig ; 14(11): 1321-1324, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37530563

ABSTRACT

The FreeStyle Libre Flash Glucose Monitoring System allows users to obtain sensor glucose values by scanning with the reader or their mobile phone. We report a case of a 59-year-old man with type 1 diabetes mellitus who developed diabetic ketoacidosis due to a sensor defect. After replacing the sensor with a new one, the glucose value shown in the device was much lower than usual, which made him consider that he was hypoglycemic. Accordingly, he reduced his insulin dose and eventually developed diabetic ketoacidosis. He was unaware of the discrepancy due to the lack of self-monitoring of his blood glucose, although he was educated to do. In sum, glucose monitoring with the FreeStyle Libre is helpful; however, it is necessary to remind the patient that a sensor defect leading to a severe complication frequently happens.


Subject(s)
Diabetes Mellitus, Type 1 , Diabetic Ketoacidosis , Humans , Male , Middle Aged , Diabetic Ketoacidosis/complications , Blood Glucose , Blood Glucose Self-Monitoring , Diabetes Mellitus, Type 1/complications , Hypoglycemic Agents/therapeutic use
12.
Biochem Biophys Res Commun ; 676: 132-140, 2023 10 08.
Article in English | MEDLINE | ID: mdl-37516030

ABSTRACT

Insulin is essential in controlling blood glucose levels, and its synthesis and secretion have been well investigated. In contrast, how insulin secretory granules (ISGs) are degraded in pancreatic beta cells remains largely unknown. To clarify the mechanism, we constructed a fluorescent reporter detecting ISG degradation, where EGFP and mCherry are tandemly conjugated to a cytoplasmic region of ZnT8, an ISG membrane-localized protein. Depletion of serum and amino acid stimulated lysosomal ISG degradation detected with the reporter. Next, with MIN6 cells expressing Cas9 and the reporter, we investigated the involvement of conventional Atg5/7-dependent autophagy to show that it is dispensable for the ISG degradation process. Finally, we performed genome-wide screening by enriching the cells lacking the ISG degradation and showed that pathways regulating autophagy are not identified. These results suggest that alternative degradation in lysosomes, instead of conventional autophagy, may be involved in ISG degradation.


Subject(s)
Insulin-Secreting Cells , Insulin , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Insulin Secretion , Membrane Proteins/metabolism , Coloring Agents/metabolism , Secretory Vesicles/metabolism , Cytoplasmic Granules/metabolism
13.
Front Physiol ; 14: 1198390, 2023.
Article in English | MEDLINE | ID: mdl-37389126

ABSTRACT

Inactivity causes insulin resistance in skeletal muscle and exacerbates various lifestyle-related diseases. We previously found that 24-h hindlimb cast immobilization (HCI) of the predominantly slow-twitch soleus muscle increased intramyocellular diacylglycerol (IMDG) and insulin resistance by activation of lipin1, and HCI after a high-fat diet (HFD) further aggravated insulin resistance. Here, we investigated the effects of HCI on the fast-twitch-predominant plantaris muscle. HCI reduced the insulin sensitivity of plantaris muscle by approximately 30%, and HCI following HFD dramatically reduced insulin sensitivity by approximately 70% without significant changes in the amount of IMDG. Insulin-stimulated phosphorylation levels of insulin receptor (IR), IR substrate-1, and Akt were reduced in parallel with the decrease in insulin sensitivity. Furthermore, tyrosine phosphatase 1B (PTP1B), a protein known to inhibit insulin action by dephosphorylating IR, was activated, and PTP1B inhibition canceled HCI-induced insulin resistance. In conclusion, HCI causes insulin resistance in the fast-twitch-predominant plantaris muscle as well as in the slow-twitch-predominant soleus muscle, and HFD potentiates these effects in both muscle types. However, the mechanism differed between soleus and plantaris muscles, since insulin resistance was mediated by the PTP1B inhibition at IR in plantaris muscle.

14.
J Diabetes Investig ; 14(6): 811-820, 2023 Jun.
Article in English | MEDLINE | ID: mdl-36942413

ABSTRACT

AIMS: For long-term management of diabetes, patients with type 2 diabetes mellitus require a high level of treatment adherence, which is associated with treatment satisfaction and their quality of life (QOL). To achieve it, patient education about diabetes self-management is essential. We routinely conduct a 7 day inpatient diabetes education program and administer the diabetes treatment-related (DTR)-QOL questionnaire to all participants, both before admission and at discharge. Here, we investigated whether our program improves QOL and post-discharge glycemic control. MATERIALS AND METHODS: This retrospective study utilized data from patients with type 2 diabetes mellitus who participated in our program between July 2017 and March 2020 and who had been treated in our outpatient department for more than 1 year. We evaluated the relationship between at admission and at discharge diabetes treatment-related quality of life scores and glycemic control after discharge. RESULTS: Data from 140 patients were analyzed in this study, which showed a significant improvement in the total, 'Anxiety and dissatisfaction with treatment', and 'Satisfaction-with-treatment' scores. A significant improvement was evident in HbA1c at 12 months after discharge. Multiple regression analysis showed that HbA1c after 12 months was independently associated with gender, duration of diabetes, and HbA1c at admission. CONCLUSIONS: Our program effectively improves quality of life and post-discharge glycemic control in patients with type 2 diabetes mellitus. It is particularly effective in patients of the male gender, with a shorter duration of diabetes mellitus and higher HbA1c at admission.


Subject(s)
Diabetes Mellitus, Type 2 , Humans , Male , Diabetes Mellitus, Type 2/therapy , Glycated Hemoglobin , Quality of Life , Blood Glucose/analysis , Patient Discharge , Inpatients , Glycemic Control , Retrospective Studies , Aftercare
15.
Cancer Immunol Immunother ; 72(7): 2347-2356, 2023 Jul.
Article in English | MEDLINE | ID: mdl-36939853

ABSTRACT

CD4+ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4+ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4+ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.


Subject(s)
Immunotherapy, Adoptive , Neoplasms , Humans , Immunotherapy, Adoptive/methods , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , CD4-Positive T-Lymphocytes , Antigens, Neoplasm
16.
Sci Rep ; 13(1): 3484, 2023 03 15.
Article in English | MEDLINE | ID: mdl-36922503

ABSTRACT

Metal homeostasis is tightly regulated in cells and organisms, and its disturbance is frequently observed in some diseases such as neurodegenerative diseases and metabolic disorders. Previous studies suggest that zinc and iron are necessary for the normal functions of pancreatic ß cells. However, the distribution of elements in normal conditions and the pathophysiological significance of dysregulated elements in the islet in diabetic conditions have remained unclear. In this study, to investigate the dynamics of elements in the pancreatic islets of a diabetic mouse model expressing human islet amyloid polypeptide (hIAPP): hIAPP transgenic (hIAPP-Tg) mice, we performed imaging analysis of elements using synchrotron scanning X-ray fluorescence microscopy and quantitative analysis of elements using inductively coupled plasma mass spectrometry. We found that in the islets, zinc significantly decreased in the early stage of diabetes, while iron gradually decreased concurrently with the increase in blood glucose levels of hIAPP-Tg mice. Notably, when zinc and/or iron were decreased in the islets of hIAPP-Tg mice, dysregulation of glucose-stimulated mitochondrial respiration was observed. Our findings may contribute to clarifying the roles of zinc and iron in islet functions under pathophysiological diabetic conditions.


Subject(s)
Diabetes Mellitus, Type 2 , Insulin-Secreting Cells , Islets of Langerhans , Humans , Mice , Animals , Islet Amyloid Polypeptide/metabolism , Zinc/metabolism , Iron/metabolism , Mice, Transgenic , Amyloid/metabolism , Islets of Langerhans/metabolism , Insulin-Secreting Cells/metabolism , Diabetes Mellitus, Type 2/metabolism
17.
Cell Chem Biol ; 30(6): 658-671.e4, 2023 06 15.
Article in English | MEDLINE | ID: mdl-36944338

ABSTRACT

Autophagy plays an essential role in preserving cellular homeostasis in pancreatic beta cells. However, the extent of autophagic flux in pancreatic islets induced in various physiological settings remains unclear. In this study, we generate transgenic mice expressing pHluorin-LC3-mCherry reporter for monitoring systemic autophagic flux by measuring the pHluorin/mCherry ratio, validating them in the starvation and insulin-deficient model. Our findings reveal that autophagic flux in pancreatic islets enhances after starvation, and suppression of the flux after short-term refeeding needs more prolonged re-starvation in islets than in the other insulin-targeted organs. Furthermore, heterogeneity of autophagic flux in pancreatic beta cells manifests under insulin resistance, and intracellular calcium influx by glucose stimulation increases more in high- than low-autophagic flux beta cells, with differential gene expression, including lipoprotein lipase. Our pHluorin-LC3-mCherry mice enable us to reveal biological insight into heterogeneity in autophagic flux in pancreatic beta cells.


Subject(s)
Insulin-Secreting Cells , Islets of Langerhans , Mice , Animals , Insulin-Secreting Cells/metabolism , Mice, Transgenic , Islets of Langerhans/metabolism , Insulin/metabolism , Autophagy/physiology
18.
Thyroid ; 33(3): 330-337, 2023 03.
Article in English | MEDLINE | ID: mdl-36565031

ABSTRACT

Background: The main molecular mechanism underlying acute suppression of iodine organification in normal thyroids after an excessive iodine load, that is, the Wolff-Chaikoff effect, is assumed to be suppression of iodine oxidation and iodothyronine synthesis. However, the mechanism underlying chronic antithyroid action of inorganic iodine in Graves' disease is not fully understood. Using a mouse model of Graves' hyperthyroidism, we examined changes in iodothyronine content and gene expression profiles in the thyroid glands after inorganic iodine loading. Materials and Methods: Graves' hyperthyroidism was induced and maintained in BALB/c mice by repeated immunizations of recombinant adenovirus expressing the human thyrotropin (TSH) receptor A-subunit. Hyperthyroid mice were left untreated (GD-C; n = 8) or treated with inorganic iodine for 12 weeks (GD-NaI; n = 8). We used unimmunized BALB/c mice as a control group (n = 10). In each mouse, serum thyroxine (T4) levels were measured with enzyme-linked immunosorbent assay (ELISA) at 4-week intervals. The intrathyroidal iodothyronine content and gene expression levels were, respectively, evaluated by mass spectrometry and RNA sequencing (RNA-seq) at the end of the experimental period. Results: Serum T4 levels in the GD-C group remained higher than in the control group, whereas those in the GD-NaI group declined to normal levels during the experimental period. Intrathyroidal triiodothyronine (T3), reverse T3 (rT3), and T4 contents in the GD-C group were higher than the control group, and rT3 and T4 were further increased in the GD-NaI group. The observed alterations in iodothyronine levels in the thyroid and sera may be explained by altered expression levels of genes for iodothyronine biosynthetic molecules, their transporter, and deiodinases. Conclusion: In this mouse model of hyperthyroidism, higher intrathyroidal accumulation of T4 and reduced gene expression data of iodothyronine transporters in the GD-NaI group suggest that chronic antithyroid action of iodine in Graves' disease involves suppression of hormone secretion.


Subject(s)
Graves Disease , Hyperthyroidism , Iodine , Humans , Thyroxine , Hyperthyroidism/genetics , Triiodothyronine , Graves Disease/genetics , Graves Disease/metabolism , Iodine/metabolism , Receptors, Thyrotropin , Triiodothyronine, Reverse , Gene Expression
19.
Juntendo Iji Zasshi ; 69(1): 42-49, 2023.
Article in English | MEDLINE | ID: mdl-38854847

ABSTRACT

Objectives: The role of autophagy in pancreatic ß cells has been reported, but the relationship between autophagy and insulin metabolism is complex and is not fully understood yet. Design: We here analyze the relationship between autophagy and insulin metabolism from a morphological aspect. Methods: We observe the morphological changes of ß cell-specific Atg7-deficient mice and Atg5-deficient MIN6 cells with electron microscopy. Results: We find that Atg7-deficient ß cells exhibit a marked expansion of the endoplasmic reticulum (ER). We also find that the inhibitory state of insulin secretion causes morphological changes in the Golgi, including ministacking and swelling. The same morphological alterations are observed when insulin secretion is suppressed in Atg5-deficient MIN6 cells. Conclusions: The defect of autophagy induces ER expansion, and inhibition of insulin secretion induces Golgi swelling, probably via ER stress and Golgi stress, respectively.

20.
Nat Commun ; 13(1): 7591, 2022 12 08.
Article in English | MEDLINE | ID: mdl-36481732

ABSTRACT

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.


Subject(s)
Anti-Bacterial Agents , Heme , Anti-Bacterial Agents/pharmacology
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